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Cloning and Expression of a Novel Endo-1, 4-β-D-glucanase Gene umcel5K and Characterization of the Translated Product

Author: TanWanXin
Tutor: FengJiaXun
School: Guangxi University
Course: Biochemistry and Molecular Biology
Keywords: uncultured microorganism metagenome endo-1,4-β-D-glucanase clone expression
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 49
Quote: 0
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Abstract


In this study, total DNA of uncultured microorganisms from the enrichment cultures of the contents of buffalo rumens was isolated . A metagenomic cosmid library that contained approximate 8×103 clones was constructed. By functional screening, a clone expressing both exoglucanase and endoglucanase activity was found from the library, and subcloning and sequencing analysis identified one gene （designated umcel5K） that contained an open reading frame （ORF） encoding a protein of 333 amino acid residues. The encoded products of the gene shared 53% identities and 68% similarities with a cellulase CelA（ABA02176） of glycosyl hydrolase family 5 from a uncultured bacterium. The ORF of umcel5K amplified by PCR was clonded into vecter pET30a（+）, and over-expressed in Escherichia coli. The Ni-NTA purified expressed product was characterized. The optimum pH of the recombinant enzyme was 4.5～5.0, and optimum temperature was 50℃for endoglucanase activity. The enzyme kinetics experiment determined that the values for Km and Vmax of Umcel5K is 3.843mg/ml and 152.4 U/ mg. Certain ions such as Fe3+、Cr2+ and Cu2+ had inhibitory effect on the activity of the enzyme, while K+、Li+ showed little influence.

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